Methods and apparatus for acoustic treatment of samples for heating and cooling

ABSTRACT

Methods and systems relate to enhancing heat transfer between a vessel wall and a sample or coupling medium during focused acoustic processing. The vessel containing the sample may include a heat exchanger on an inner surface and/or an outer surface of the vessel that can have any suitable shape or dimension that increases the surface area of the vessel wall. In some embodiments, heat exchanger features may disrupt a boundary layer of a liquid sample at the vessel wall during focused acoustic processing. Accordingly, the temperature of the liquid sample can be appropriately controlled. In some cases, heating and/or cooling of the liquid sample may be performed efficiently. In an embodiment, a liquid sample may be heated at a rate of at least about 25 degrees C. per ml per minute.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/368,410, filed Jul. 28, 2010, which is hereby incorporated byreference in its entirety.

BACKGROUND

1. Field of Invention

Aspects described herein relate to acoustic treatment of samples, suchas liquid material contained in a well of a microtiter plate or othersimilar vessel. In some cases, acoustic treatment of a sample mayinvolve enhancing heat transfer between the vessel wall and the sample,such as through the disruption of a boundary layer at a vessel wall.

2. Related Art

Analytical techniques for biological and chemical samples often requirean extreme physicochemical preparatory step to enable the desiredanalysis to be fully achieved. For example, extraction/digestion ofherbicides and pesticides from plant tissue may require organic solvents(e.g., alcohols) and elevated temperatures (e.g., 50 degrees C.). Thisrequirement to elevate the temperature of a sample to aid extraction ofa desired component or constituent of a sample is a commonly usedtechnique. For example, many environmental sample analysis techniquesrequire thermal energy to aid extraction. Another area whereby thermalenergy is utilized to aid sample preparation is in microbial analysis;difficult cell wall disruption is aided by thermal energy.

Typically, transfer of thermal energy for such processes is achievedwhen heat is transferred from an area at higher temperature to a regionof the sample at a lower temperature. For a biological or chemicalsample contained in an isolated environment within a sample vessel, suchheat transfer occurs by convection-based diffusion processes (Brownianmotion and eddy diffusion) and advective fluid bulk transport(larger-scale current flow) processes. This is inherently a slow processand is exacerbated as the sample volume is increased (i.e., where thevolume increases at a greater rate than the contact thermal surfacearea).

For example, a standard extraction/digestion process often used withsample slurries employs a combination of a stirring magnetic field torotate a magnetic stir-bar in the sample fluid contained in a glassvessel and a hot plate to heat the vessel. The stir-bar imparts largescale currents, which ideally uniformly transfer the heat at the vesselwall to the entire fluid. An alternative means to transfer thermalenergy is to use focused microwave techniques for biological andchemical processing. Indeed, even with closed vessel microwave heatexchange techniques, a magnetic stir-bar is utilized to impart largescale currents in the sample to be processed.

SUMMARY

In accordance with aspects of the invention, control of acoustic energyenables both heating of the vessel wall to heat a sample and disruptionof a boundary layer of a sample liquid at the vessel wall to enhanceheat transfer between the vessel wall and the sample. In other words,acoustic peak positive and peak negative zones may impart fluid movementfor large-scale current formation as well as heating of the vessel wall.Heating of the vessel wall may be caused by an intrinsic acousticimpedance mismatch between materials (e.g., between the vessel wall anda surrounding acoustic coupling medium) such that a portion of theacoustic compression/rarefaction energy is absorbed by the vessel wall.The acoustic energy may also cause portions of the sample located at thevessel wall to flow, thereby enhancing heat transfer from the vesselwall to the sample. As a result, both mixing and heating of the samplecan be performed without physically contacting the sample with anystructure aside from the vessel. Also, some processes may benefit fromexposing the sample to both elevated pressures and temperatures (i.e.,pressures and temperatures above ambient). Aspects described herein maybe useful with such processes since the sample may be both thermallyheated as well as exposed to elevated pressures by way of cavitation orother conditions caused by the acoustic energy.

In one aspect of the invention, a method for acoustic treatment of asample contained in a vessel includes providing a vessel containing aliquid sample where the vessel has a wall in contact with the liquidsample. The vessel wall may include a heat exchanger on an inner surfacethat is in contact with the liquid sample and/or a heat exchanger on anouter surface of the wall that is in contact with an acoustic couplingmedium. The heat exchanger on the inner and/or outer surfaces may take avariety of forms, such as fins, bumps, grooves and/or other physicalfeatures that help increase a surface area of the vessel wall in contactwith the sample or a coupling medium. The heat exchanger features at theinner surface of the vessel may also, or alternately, be arranged tohelp disrupt a boundary layer of the liquid sample at the vessel wall,e.g., to help induce large scale mixing or other flow of the sample toenhance heat transfer. Thus, the method may further include applyingacoustic energy from an acoustic energy source to the liquid sample tocause movement of portions of the liquid sample near the vessel wall,and using a heat exchanger on the inner surface of the vessel wall tointeract with moving portions of the liquid sample and disrupt aboundary layer of the liquid sample at the vessel wall, such thatdisruption of the boundary layer enhances heat transfer between thevessel wall and the liquid sample.

Heat transfer between the vessel wall and the sample may be used to heator cool the sample. For example, simultaneous with disrupting theboundary layer of the sample at the vessel wall, acoustic energy may beapplied from the acoustic energy source to the vessel wall to heat thevessel wall and increase the vessel wall's temperature above atemperature of the liquid sample. As will be understood, heating thevessel wall causes heat transfer from the vessel wall to the liquidsample to raise the temperature of the liquid sample. In someembodiments, the temperature of the sample may be raised above atemperature of a coupling medium in contact with an exterior of thevessel. The temperature of the sample may be detected, e.g., by aninfrared detector, and the acoustic energy controlled so as to maintainthe sample temperature constant, or to vary the temperature of thesample.

In other embodiments, the sample may be cooled. For example, atemperature of the vessel wall may be below a temperature of the liquidsample, and the boundary layer may be disrupted to cause heat transferfrom the liquid sample to the vessel wall so as to lower a temperatureof the liquid sample. The vessel wall may be cooled in any suitable way,such as by transferring heat from the vessel wall to a coupling mediumin contact with the vessel wall. In one embodiment, the coupling mediummay be liquid water, although other liquid, solid and semi-solidmaterials may be used to couple acoustic energy to the vessel.

When heating or cooling the sample by transfer of heat between thevessel wall and a coupling medium, a heat exchanger at the outer surfaceof the vessel wall may be employed. The heat exchanger may includephysical features on the vessel wall, such as fins, ribs, grooves, ametal element or other relatively highly thermally conductive member,and so on. Disruption of a boundary layer of the liquid sample at thevessel wall as discussed above may also assist in enhancing heattransfer between the sample and the vessel wall.

In another aspect of the invention, a method for acoustic treatment of asample contained in a vessel includes providing a vessel containing aliquid sample where the vessel has a wall with an inner surface incontact with the liquid sample. A coupling medium, which may be a singlematerial such as liquid water, or two or more materials, may be providedin contact with an outer surface of the vessel such that the couplingmedium may transmit acoustic energy to the vessel. Acoustic energy maybe applied from an acoustic energy source through the coupling medium tothe vessel wall to heat the vessel wall and increase the vessel wall'stemperature above a temperature of the liquid sample. As discussedabove, for some embodiments, the acoustic energy may take advantage ofimpedance mismatches between the vessel wall and the coupling mediumand/or the sample to heat the vessel wall. Simultaneous with applyingacoustic energy to heat the vessel wall, acoustic energy may be appliedfrom the acoustic energy source to the liquid sample to disrupt aboundary layer of the liquid sample at the vessel wall so as to enhanceheat transfer from the vessel wall to the liquid sample and to raise thetemperature of the liquid sample above a temperature of the couplingmedium. In one embodiment, heating of the liquid sample may be performedat a rate of at least about 25 degrees C. per ml per minute. This rapidheating capability is unknown in the prior art, and may be enabled bythe use of a heat exchanger or other element to disrupt the boundarylayer of the liquid sample at the vessel wall. That is, by physicallydisrupting the boundary layer, more effective sample flow or othermovement may be caused, which results in more efficient heat transfer.

These and other aspects of the invention will be apparent from thefollowing description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the invention are shown and described with reference toillustrative embodiments and the following drawings, in which likenumerals reference like elements, and wherein:

FIG. 1 shows a schematic diagram of an acoustic treatment system inaccordance with an aspect of the invention;

FIG. 2 shows a schematic cross sectional diagram of a vessel having aheat exchanger element at an inner surface in a illustrative embodiment;and

FIG. 3 shows a vessel having a heat exchanger at an outer surface in anillustrative embodiment.

DETAILED DESCRIPTION

Although aspects of the invention are described with reference toembodiments in which acoustic energy is used to heat and/or cool asample, the sample may be subjected to other treatments or otherprocesses by the acoustic energy. For example, the acoustic energy mayalso be suitable, or be adjusted, to cause other effects in the liquid,such as fluidizing the sample, mixing the sample, stirring the sample,catalyzing the sample, disrupting the sample (such as shearing orfragmenting DNA molecules or other compounds, lysing cells, etc.),permeabilizing a component of the sample, enhancing a reaction in thesample (such as binding of material to the material supports), causingcrystal growth in the sample (e.g., by nucleating crystal growth sitesand/or enhancing the rate of crystal growth), preparing formulations(e.g., suspensions and/or emulsions suitable for therapeutic use),causing flow in a conduit, and/or sterilizing the sample. Thus, theacoustic energy may be used for other purposes than merely heatingand/or cooling a sample. In other embodiments, the acoustic energy mayfacilitate chemical or other reactions in the liquid, which generate anend product that is to be separated from the liquid and other substancesin the liquid, e.g., using beads or other structures that bind to theend product to be separated. In addition, under the applied acousticenergy, a controlled active turbulent regime may exist, whereby thecollision frequency between binding partners in the sample and on beadsor other structures is increased. This actively controlled turbulencemay accelerate desired processes, as opposed to passive diffusiondominated processes of paramagnetic or other currently available beadproducts.

The samples can be treated in any suitable vessel provided that thevessel in at least some embodiments includes one or more aspects of theinvention. Vessels can be sealed for the duration of the treatment toprevent contamination of the sample or of an environment outside of thevessel, and arrays of vessels can be used for processing large numbersof samples. These arrays can be arranged in one or more high throughputconfigurations. Examples include microtiter plates, optionally with atemporary sealing layer to close the wells, blister packs, similar tothose used to package pharmaceuticals such as pills and capsules, andarrays of polymeric bubbles, similar to bubble wrap, preferably with asimilar spacing to typical microtiter wells. Vessels containing thesamples can be sealed during the processing, and hence can be sterilethroughout, or after, the procedure. Moreover, the use of focusedultrasound allows the samples in the vessels to be processed, includingprocessing by stirring, without contacting the samples, even when thevessels are not sealed. Thus, a sample vessel can be a membrane pouch,thermopolymer well, polymeric pouch, hydrophobic membrane, microtiterplate, microtiter well, test tube, centrifuge tube, microfuge tube,ampoule, capsule, bottle, beaker, flask, and/or capillary tube.

Any suitable sample material can be included in a vessel, and the samplemay include any suitable combination of a liquid (such as a solvent), asolid material (such as pieces of bone, tissue or plant materials), adissolved material (such as a salt) and so on. Some example materialsthat may be included in a sample are DNA, RNA, nucleic acids, or othergenetic material, antibodies, receptors and/or ligands associated withcellular functions, proteins, polymers, amino acid monomers, an aminoacid chain, enzymes, nucleic acid monomers or chains, saccharides orpolysaccharides, lipids, organic or inorganic molecules, vectors,plasmids, pharmaceutical agents, compositions suitable for crystalgrowth, prions, bacteria, and/or viruses. This is not intended to be anexhaustive list, but rather to provide a few examples of sample materialthat may be used with aspects of the invention.

FIG. 1 shows a schematic diagram of an acoustic treatment system 100that incorporates one or more aspects of the invention. In thisillustrative embodiment, the system 100 includes an acoustic transducer1 that is arranged to emit sonic energy through a coupling medium 2,such as a liquid (e.g., water, organic solution, etc.) held in acontainer 3 or a solid material (e.g., elastomeric material, gel,silicone, rubber, etc.) in contact with the transducer 1, and form afocal zone 11 of acoustic energy near or at a vessel 4. The acousticenergy at the focal zone may be suitable to cause heating, mixing,cavitation or other effects in a sample 6 located in the vessel 4.Cavitation or other conditions induced by acoustic energy at the focalzone may create localized relatively high pressure (and/or low pressure)conditions that may be useful in enhancing reactions in samplematerials. The vessel 4 may have an interior volume of any suitablesize, e.g., between 1 μL and 100 milliliters.

A controller 5 may provide suitable control signals to the transducer 1to generate desired acoustic energy, and control the relative positionof the vessel 4 and the transducer 1 (e.g., in 3 dimensions) so that thesample 6 in the vessel 4 may be suitably positioned relative to thefocal zone 11. Further details regarding an illustrative embodiment foran acoustic treatment system 100 are provided below, and in U.S. Pat.No. 6,948,843, which is incorporated herein by reference in itsentirety. For example, the focal zone 11 may have a spherical, egg-like,or elongated rod-like shape, may include two or more focal zones orfocal lines (e.g., focal zones with high aspect ratios), and so on.

In accordance with an aspect of the invention, the vessel 4 may includeone or more heat exchanger features that are located in contact with thesample 6 and/or in contact with the coupling medium 2. When used at theinner surface of the vessel, the heat exchanger features can enablerapid heating of the sample, e.g., by enabling the disruption of aboundary layer of the sample at the vessel wall. Generally, the boundarylayer may be considered herein as a layer of fluid immediately adjacentto a solid surface where certain effects (e.g., due to viscosity)arising from the presence of the solid surface play a non-negligiblerole. For example, a boundary layer may be a fluid layer adjacent avessel wall that, in the absence of acoustic mixing/agitation, remainsrelatively stagnant, substantially does not transfer heat between thevessel wall and the fluid by convection, and instead transfers heatbetween the vessel wall and the fluid by radiation and/or conduction.When the boundary layer is sufficiently disrupted (e.g., by focusedacoustic treatment), convective heat transfer between the vessel walland the fluid occurs more freely. In some embodiments, for a vessellacking a heat exchanger or similar feature at the inner wall, theboundary layer of sample at the vessel wall may remain undisturbed,essentially forming a region that behaves as a blanket of insulationthat forces heat transfer by radiation or conduction processes only. Incontrast, the heat exchanger features in accordance with an aspect ofthe invention at the inner wall of a vessel allow the acoustic energy todisrupt this boundary layer, thereby enabling convective heat transferin addition to radiation and conduction modes.

Disruption of the boundary layer enabled by a heat exchanger featurecreates large scale flow at the vessel wall and thus permits rapid heattransfer between the sample and the vessel. In cases where the vesselwall is at a higher temperature than the sample, the sample can beheated quickly, particularly where the vessel wall is being heated byacoustic energy. FIG. 2 shows a cross sectional view of a vessel 4 thatincludes heat exchanger features 7 in the form of an array of raisedareas on the inner surface of the vessel wall. In this embodiment, theraised areas are arranged in a regular pattern of individual bumps thatextends around the inner periphery of the vessel 4 and along at leastpart of the length of the vessel 4. These bumps 7 cause turbulence inflow occurring near the vessel wall, thereby breaking up a relativelystagnant boundary layer that might otherwise form. This breakup inducesimproved convective heat flow, allowing the sample to be heated orcooled more rapidly. The inventor has found that these features canenable extremely rapid heating of at least 25 degrees C. per milliliterof liquid per minute (degrees C. per ml per min). Heating this rapid isunknown in prior art applications that do not involve focused acousticsand one or more aspects of the invention.

The heat exchanger features 7 can be formed in any suitable way such asby molding, thermoforming, machining, etching, applying with anadhesive, and so on. For example, the heat exchanger 7 may be formed aspart of a sleeve that is inserted into the vessel and bonded (e.g., withan adhesive, application of pressure, mechanical fit, etc.) to the innerwall. In another embodiment, the heat exchanger 7 may be moldedintegrally with the vessel wall. In addition, the shape, size andarrangement of heat exchanger features may be arranged in any suitableway. In the embodiment of FIG. 2, the heat exchanger features have amesa-type shape, but may be arranged as fins, rods, smooth bumps,grooves, holes, pits, tabs, and others. Also, in this embodiment, theraised areas have a size of about 1 sq. millimeter, a height of about100 micrometers and are separated from each other by a spacing of about3 millimeters, but other sizings and spacings are possible. For example,the size, shape and/or space between features may be varied according toa frequency or set of frequencies used to treat the sample 6. In oneembodiment, a variety of differently sized and spaced features may beused so that different sets of features may selectively interact withacoustic energy within a certain frequency range. That is, features of afirst size/shape/spacing may interact most strongly with acoustic energyin a first frequency range, features of a second size/shape/spacing mayinteract most strongly with acoustic energy in a second frequency range,and so on. As a result, the different features may be activated atdifferent times, e.g., if the sample 6 is treated with a sweep ofvarying frequency acoustic energy.

Heat exchanger features may be formed as positive features that extendfrom the vessel wall into the vessel and/or negative features thatextend into the vessel wall. Different types of heat exchanger featuresmay be used together, such as an array of bumps combined with an arrayof grooves. In short, the heat exchanger features may be arranged so asto maximize boundary layer disruption for one or more particularapplications. Since different applications may involve differentmaterials in the sample and/or different sample viscosities, the heatexchanger features may be arranged to work best with a specific sampleviscosity range and/or particle sizes.

As noted above, a vessel 4 may include heat exchanger features at aninner surface of the vessel wall or at an outer surface of the wall.FIG. 3 shows another embodiment in which a vessel 4 includes heatexchanger features 8 on an exterior of the vessel. In this embodiment,the heat exchanger features 8 are arranged as longitudinal fins thatextend along a length of the vessel. In contrast to the heat exchangerfeatures 7 at the interior of the vessel, the heat exchanger features 8on the exterior of the vessel need not necessarily function to disrupt aboundary layer of a coupling medium or other liquid at the exterior ofthe vessel. Instead, heat exchanger features 8 at the vessel exteriormay function to help increase surface area and heat transfer to aliquid, solid or semi-solid coupling medium (such as water, a silicamaterial, and/or a silicone rubber). By exchanging heat with thecoupling medium, the vessel can be heated and/or cooled so long as thereis a temperature difference between the vessel and the coupling medium.As discussed above, heat transfer between the vessel and the sample canheat and cool the sample, and thus the coupling medium can be used tocool and/or heat the sample in certain circumstances. By providing heatexchanger features 8 on the vessel exterior, heat transfer between thevessel and the coupling medium can be better controlled, allowing formore accurate and efficient thermal cycling treatments of the sample tobe performed.

As with the heat exchanger features 7 at the vessel interior, the heatexchanger features 8 can be arranged in any suitable way, with anysuitable size, shape and/or configuration. Although the FIG. 3embodiment shows the heat exchanger features 8 in the form oflongitudinal fins, the heat exchanger features may include bumps,grooves, pits, circumferential or spiral fins (e.g., having awasher-like shape), plates, mushroom-like structures, studs, and others.The heat exchanger features 8 may be formed unitarily with the vessel(e.g., molded into the vessel wall), attached to the vessel wall (e.g.,by an adhesive, sonic welding, or other) and so on. For example, in oneembodiment, heat exchanger features 8 may be formed on a sleeve (such asa highly conductive metal sheath) that is slid over the vessel andbonded in place. In other embodiments, the heat exchanger features maybe attached to the vessel using an interference or friction fit, such asmetallic washer-shaped elements that are pressed onto the vessel wallsuch that the hole of the washer element fits tightly to the vesselouter surface. The heat exchanger may have portions that extend throughthe vessel wall, such as metallic stud elements that extend from outsidethe vessel wall, through the wall and into the vessel interior. In oneembodiment, such heat exchanger features may be molded with a plasticmaterial to make the vessel. For example, the metallic studs may bemounted in a mold and molten plastic injected so that the studs areformed integrally with the vessel and extend from inside to outside ofthe vessel. In one embodiment, such studs or similar elements may formboth heat exchanger features at the inner surface of the vessel wall andheat exchanger features at the outer surface of the vessel wall.

When using a vessel in accordance with aspects of the invention, thetemperature of the external environment of the sample vessel (e.g., thecoupling medium) may be below the temperature of the sample during atreatment process. This arrangement enables the sample to beintermittently elevated in temperature for a desired process. Forexample, a sample in a polypropylene plastic tube and cap may initiallybe at 4 degree C. with the tube placed in a 96 tube rack. A focusedacoustic field may be directed to the sample, which is contained in oneof the tubes in the rack. During an acoustic dose, the internaltemperature of the sample may be increased to 50 degree C. withinseconds (e.g., less than 10 seconds). If the sample is initially frozen,this thermal energy may be used to quickly thaw the frozen sample. Inaccordance with an aspect of the invention, only one of the samples inthe rack may be thawed while other samples remain frozen. This would beof benefit if the rack of samples (e.g., 96 tubes) were at −20 degreeC., but only one sample was required to be thawed for processing. Rapidheating enabled by aspects of the invention has been found by theinventor to be significantly faster than other prior processes. Forexample, compare a process of thawing a biological fluidic sample (e.g.,serum) that is initially at (−70) degrees C. in which the sample isplaced in a water bath at 20 degree C. to a process in accordance withaspects of the invention. Simply placing the sample in a 20 degree C.water bath typically requires several minutes before the sample reachesa temperature at 20 degree C. However, with an applied acoustic fieldand heat exchanger elements used with the vessel, a sample thaw mayoccur within 10 seconds even with the coupling medium at a relativelylower temperature of 5 degrees C.

In other embodiments, the temperature of the external environment of thesample vessel (e.g., the coupling medium temperature) may be elevatedabove the sample temperature, at least initially. In this situation, arise in temperature of the sample, if desired, may be furtheraccelerated. For example, a −70 degree C. frozen sample may be placedinto a water bath of 20 degrees C. and an acoustic dose applied to thevessel. As the vessel wall is heated by the acoustic energy, the fluidmotion turbulence generated by the acoustic energy and a heat exchangerin the vessel further aids the heat transfer from the vessel wall andthe coupling medium to the sample. Similar is true where the sample isto be cooled where the sample temperature is higher than the couplingmedium. Thus, the heat transfer process may be accelerated for bothheating and cooling of the sample by appropriate setting of the couplingmedium temperature. This may be of value in thermal cycling ofbiological processes, such as thermo-stabile enzymes.

The controller 5 may be arranged to control the transducer 1 in anysuitable way, e.g., generate a variety of alternating voltage waveformsto drive the transducer 1. For instance, a high power “treatment”interval consisting of about 5 to 1,000 sine waves, for example, at 1.1MHz, may be followed by a low power “convection mixing” intervalconsisting of about 1,000 to 1,000,000 sine waves, for example, at thesame frequency. (Although there is a short time period separationbetween treatment and mixing intervals, the intervals are referred toherein as occurring simultaneously, i.e., acoustic energy to causeheating is said to be applied simultaneously with acoustic energy tocause mixing.) It is during the convective mixing interval that heatexchanger elements in the vessel may maximally assist in disrupting theboundary layer at the vessel wall. “Dead times” or quiescent intervalsof about 100 microseconds to 100 milliseconds, for example, may beprogrammed to occur between the treatment and convection mixingintervals. Also, the focal zone 11 may be arranged in any suitable way,e.g., may be small relative to the dimensions of the vessel 4 to avoidheating of the treatment vessel during some intervals, or may be largerthan the vessel 4. In one embodiment, the focal zone 11 may have a widthof about 2 cm or less, a height of about 6 cm or less and a length of 5cm or more. In another embodiment, the focal zone 11 may have anellipsoidal shape, with a width or diameter of about 2 cm or less and alength of about 6 cm or less.

Acoustic energy in the focal zone 11 may generate a shock wave that ischaracterized by a rapid shock front with a positive peak pressure inthe range of about 15 MPa, and a negative peak pressure in the range ofabout negative 5 MPa. This waveform may be of about a few microsecondsduration, such as about 5 microseconds. If the negative peak is greaterthan about 1 MPa, cavitation bubbles may form in liquid in the sample.Cavitation bubble formation may also be dependent upon the surroundingmedium 2, the vessel material, or other features. For example, glycerolis a cavitation inhibitive medium, whereas liquid water is a cavitationpromotive medium. The collapse of cavitation bubbles may form“microjets” and turbulence that impinge on the surrounding material.These cavitation bubbles may contribute to sample liquid movement duringa treatment. Moreover, the localized high and low pressure regions mayexpose portions of the sample to suitable pressures and temperaturesthat are useful for causing some chemical reactions or other results.

In the embodiments shown, the acoustic energy is transmitted from thetransducer 1 to the vessel 4 through a medium 2, such as water. However,other media or combinations of media may be used, such as a solid orsemi-solid material and others. For example, the transducer 1 may bemated to a solid silica-based material that conducts acoustic energytoward the sample vessel. A semi-solid material, such as a siliconerubber or gel, may be used to couple the silica material to the vessel.The water bath or other acoustic coupling media (e.g., silicone rubber)may be at room temperature and the sample may be contained in a vesselwhich readily transfers heat (e.g., borosilicate glass), but allows theacoustic energy to couple directly with the internal sample for heattransfer. For example, a 20% glycerol sample will be more sensitive toacoustic energy-mediated temperature elevation than a 2% glycerolsample. In this embodiment, the vessel wall may be more transparent toacoustic energy, and thereby resulting in the sample or sampleconstituents absorbing the acoustic energy and impart thermal energytransfer directly to the sample. An example of a vessel wall materialwith desired acoustic properties is the low density, transparentthermoplastic polymer of methylpentene monomer units (polymethylpenteneor TPX).

The geometry and material choice of the vessel wall may also affect theperformance of the non-contact, acoustic treatment. In addition, theinternal vessel volume and the ratio of sample to headspace will alsoaffect the performance of the device. For example, a 1.5 milliliterconical polypropylene tube with 1.0 milliliter of sample when placedinto a 0.5 MHz focused acoustic field converging on the cone of the tubewould enable the internal, starting temperature of 20 degree C.(external water bath temperature) to be elevated to 90 degree C. in lessthan 120 seconds at a high acoustic dose. The temperature may quickly belowered to 20 degree C. with a lower acoustic dose to dissipate thethermal energy.

Many types of acoustic systems may be used to generate the appropriatewave-train to impart the thermal energy transfer. For example, anunfocused acoustic source (15 kHz) directed toward the vessel wouldresult in the vessel wall temperature rise, which would thereby heat theinternal fluidic sample. Alternatively, a focused acoustic source (e.g.,0.5 MHz) may also be used. In both situations, a feedback loop algorithmmay be utilized to automate and control the process, e.g., monitoringthe external temperature of the vessel wall may indirectly indicate theappropriate dose to be applied to the sample. In one embodiment, theapparatus may have an external non-contact infrared meter monitoring theexternal temperature of the sample vessel. For example, during anacoustic extraction dose, the vessel wall temperature will increase andthe fluidic sample will be turbulent. The turbulence will effectivelytransfer the temperature throughout the sample and thereby enableexternal thermal measurements to provide an indication of internaltemperature. This is particularly valid if the sample is thoroughlywashing the internal walls of the vessel during the acoustic dose. Thus,a heat exchanger 7 at the vessel inner surface may enable more accuratetemperature measurement of the sample.

Many biological and other materials can be treated according to aspectsof the invention. For example, such materials for treatment include,without limitation, growing plant tissue such as root tips, meristem,and callus, bone, yeast and other microorganisms with tough cell walls;bacterial cells and/or cultures on agar plates or in growth media, stemor blood cells, hybridomas and other cells from immortalized cell lines,and embryos. Additionally, other biological materials, such as serum andprotein preparations, can be treated with the processes of theinvention, including sterilization.

Many binding reactions can be enhanced with treatments in accordancewith aspects of the present disclosure. Binding reactions involvebinding together two or more molecules, for example, two nucleic acidmolecules, by hybridization or other non-covalent binding. Bindingreactions are found, for example, in an assay to detect binding, such asa specific staining reaction, in a reaction such as the polymerase chainreaction where one nucleotide molecule is a primer and the other is asubstrate molecule to be replicated, or in a binding interactioninvolving an antibody and the molecule it binds, such as an immunoassay.Reactions also can involve binding of a substrate and a ligand. Forexample, a substrate such as an antibody or receptor can be immobilizedon a support surface, for use in purification or separation techniquesof epitopes, ligands, and other molecules.

In certain embodiments, temperature, mixing, or both can be controlledwith ultrasonic energy to enhance a chemical reaction. For example, theassociation rate between a ligand present in a sample to be treated anda binding partner on a bead or other support in the sample can beaccelerated. In another example, an assay is performed where temperatureis maintained and mixing is increased to improve association of two ormore molecules compared to ambient conditions. It is possible to combinethe various aspects of the process described herein by first subjectinga mixture to heat and mixing in order to separate a ligand or analyte inthe mixture from endogenous binding partners in the mixture. Thetemperature, mixing, or both, are changed from the initial condition toenhance ligand complex formation with a binding partner relative toligand/endogenous binding partner complex formation at ambienttemperature and mixing. Generally, the second temperature and/or mixingconditions are intermediate between ambient conditions and theconditions used in the first separating step above. At the secondtemperature and mixing condition, the separated ligand may be reactedwith the binding partner.

Focused sonic fields can be used to enhance separations. As notedelsewhere, sonic foci can be used to diminish or eliminate wall effectsin fluid flow, which is an important element of many separationprocesses, such as chromatography including gas chromatography, sizeexclusion chromatography, ion exchange chromatography, and other knownforms, including filed-flow fractionation. The ability to remotelymodulate and/or reduce or eliminate the velocity and concentrationgradients of a flowing stream is applicable in a wide variety ofsituations, such as those described in relation to FIG. 2.

Sonic energy fields can be used to enhance reaction rates in a viscousmedium, by providing remote stirring on a micro scale with minimalheating and/or sample damage. Heat exchanger features in a vessel may beuseful in promoting micro and larger scale stirring whether with orwithout significant heat transfer. Likewise, any bimolecular(second-order) reaction where the reactants are not mixed at a molecularscale, each homogenously dissolved in the same phase, potentially can beaccelerated by sonic stirring. At scales larger than a few nanometers,convection or stirring can potentially minimize local concentrationgradients and thereby increase the rate of reaction. This effect can beimportant when both reactants are macromolecules, such as an antibodyand a large target for the antibody, such as a cell, since theirdiffusion rates are relatively slow and desorption rates may not besignificant.

These advantages may be realized inexpensively on multiple samples in anarray, such as a microtiter plate. The use of remote sonic mixingprovides a substantially instantaneous start time to a reaction when thesample and analytical reagents are of different densities, because insmall vessels, such as the wells of a 96 or 384 well plate, littlemixing will occur when a normal-density sample (about 1 g/cc) is layeredover a higher-density reagent mixture. Remote sonic mixing can start thereaction at a defined time and control its rate, when required. Steppingand dithering functions may allow multiple readings of the progress ofthe reaction to be made. The mode of detecting reaction conditions canbe varied between samples if necessary. In fact, observations bymultiple monitoring techniques, such as the use of differing opticaltechniques, can be used on the same sample at each pass through one ormore detection regions.

By focusing and positioning sonic energy near a wall of a vessel, e.g.,at heat exchanger features, many local differences in the distributionof materials within a sample and/or spatially-derived reaction barriers,particularly in reactive and flowing systems, can be reduced to theminimum delays required for microscopic diffusion. Put differently,enhanced mixing can be obtained in situations where imperfect mixing iscommon. For example, if sonic energy is focused in, on, or near the wallof the vessel, near the fluid/wall boundary, then local turbulence canbe obtained without a high rate of bulk fluid flow. Excitation of thenear-wall fluid in a continuous, scanned, or burst mode can lead torapid homogenization of the fluid composition just downstream of theexcited zone, e.g., a short distance away from a boundary layer at aheat exchanger feature.

This effect is useful in several areas, including chromatography; fluidflow in analytical devices, such as clinical chemistry analyzers; andconversion of the fluid in a pipeline from one grade or type to another.Since most of the effect occurs in a narrow zone, only a narrow zone ofthe conduit typically needs to be sonically excited, and only the narrowzone need include heat exchanger features at the vessel wall. Forexample, in some applications, the focal zone of the sonic energy can bethe region closest to a valve or other device which initiates the switchof composition. In any of these applications, feedback control can bebased on local temperature rise in the fluid at a point near to ordownstream of the excitation region.

Focused acoustics and heat exchanger features in accordance with aspectsof the present disclosure may be useful for preparing formulationshaving a narrow particle size distribution. Such formulations mayinclude suspensions and/or emulsions having particles that are submicronin size and may have applications for therapeutic use, such as deliverysystems for bioactive agents (e.g., liposomes, micelles, etc.).Controlling heat transfer in a focused acoustic processing system usingheat exchanger features contemplated may enhance the ability to suitablyprepare formulations in an advantageous manner (e.g., repeatable, shortprocessing times, high yield, etc.).

In some embodiments, enhancing heat transfer between the wall of aprocessing vessel and a fluid upon focused acoustic treatment may alsobe useful for initiating (e.g., forming nucleation sites) and augmenting(nano)crystalline growth. For example, crystalline particles may beformed as a suspension of particles (e.g., submicron in size) in aliquid solution. In some cases, though not required, (nano)crystallineparticles prepared in accordance with aspects described herein may beuseful for therapeutic delivery of bioactive agents.

While there has been described herein what are considered to beexemplary and preferred embodiments of the invention, othermodifications and alternatives of the inventions will be apparent tothose skilled in the art from the teachings herein. All suchmodifications and alternatives are considered to be within the scope ofthe invention.

1. A method for acoustic treatment of a sample contained in a vessel,comprising: providing a vessel containing a liquid sample, the vesselhaving a wall in contact with the liquid sample, the wall including aheat exchanger on an inner surface that is in contact with the liquidsample; applying acoustic energy from an acoustic energy source to theliquid sample to cause movement of portions of the liquid sample nearthe vessel wall; and using the heat exchanger on the inner surface ofthe vessel wall to interact with moving portions of the liquid sampleand disrupt a boundary layer of the liquid sample at the vessel wall,disruption of the boundary layer enhancing heat transfer between thevessel wall and the liquid sample.
 2. The method of claim 1, furthercomprising: simultaneous with disrupting the boundary layer, applyingacoustic energy from the acoustic energy source to the vessel wall toheat the vessel wall and increase the vessel wall's temperature above atemperature of the liquid sample.
 3. The method of claim 2, whereinheating the vessel wall causes heat transfer from the vessel wall to theliquid sample to raise the temperature of the liquid sample above atemperature of a coupling medium in contact with an exterior of thevessel.
 4. The method of claim 1, wherein a temperature of the vesselwall is below a temperature of the liquid sample, and disrupting theboundary layer causes heat transfer from the liquid sample to the vesselwall so as to lower a temperature of the liquid sample.
 5. The method ofclaim 1, wherein the heat exchanger includes a plurality of raised areasand/or grooves on the vessel wall.
 6. The method of claim 1, wherein theheat exchanger extends around an entire internal periphery of the vesselwall and extends along at least a portion of a length of the vessel. 7.The method of claim 1, wherein the heat exchanger includes features thatare molded integrally with the vessel wall.
 8. The method of claim 1,wherein the vessel wall includes a heat exchanger on an outer surface tointeract with an environment outside of the vessel.
 9. The method ofclaim 1, simultaneous with disrupting the boundary layer, applyingacoustic energy from the acoustic energy source to the vessel wall toheat the vessel wall and heat the liquid sample at a rate of at leastabout 25 degrees C. per ml per minute.
 10. A method for acoustictreatment of a sample contained in a vessel, comprising: providing avessel containing a liquid sample, the vessel having a wall in contactwith the liquid sample, the wall including a heat exchanger on an outersurface; providing a coupling medium in contact with the heat exchangerof the vessel, the coupling medium having a temperature that isdifferent from a temperature of the liquid sample; and disrupting aboundary layer between the liquid sample and the wall by transmittingacoustic energy through the coupling medium and to the vessel and liquidsample to cause movement of portions of the liquid sample, disruption ofthe boundary layer enhancing heat transfer between the liquid sample andthe vessel wall.
 11. The method of claim 10, wherein the heat exchangerincludes a plurality of raised areas and/or grooves on the outer surfaceof the vessel wall.
 12. The method of claim 10, further comprising:simultaneous with disrupting the boundary layer, applying acousticenergy from the acoustic energy source to the vessel wall to heat thevessel wall and increase the vessel wall's temperature above atemperature of the liquid sample.
 13. The method of claim 12, whereinheating the vessel wall causes heat transfer from the vessel wall to theliquid sample to raise the temperature of the liquid sample above thetemperature of the coupling medium.
 14. The method of claim 10, whereina temperature of the vessel wall is below a temperature of the liquidsample, and disrupting the boundary layer causes heat transfer from theliquid sample to the vessel wall so as to lower a temperature of theliquid sample.
 15. The method of claim 10, wherein the vessel is one ofa plurality of vessels in a multi-well plate.
 16. The method of claim10, wherein the heat exchanger extends around an entire externalperiphery of the vessel wall and extends along at least a portion of alength of the vessel.
 17. The method of claim 10, wherein the heatexchanger includes features that are molded integrally with the vesselwall.
 18. The method of claim 10, wherein the vessel wall includes aheat exchanger on an inner surface to interact with the liquid sample.19. The method of claim 10, simultaneous with disrupting the boundarylayer, applying acoustic energy from the acoustic energy source to thevessel wall to heat the vessel wall and heat the liquid sample at a rateof at least about 25 degrees C. per ml per minute.
 20. The method ofclaim 10, wherein the temperature of the coupling medium is lower than atemperature of the liquid sample, and the step of disrupting causes atemperature of the liquid sample to be reduced.
 21. A method foracoustic treatment of a sample contained in a vessel, comprising:providing a vessel containing a liquid sample, the vessel having a wallwith an inner surface in contact with the liquid sample; providing acoupling medium in contact with an outer surface of the vessel; applyingacoustic energy from an acoustic energy source through the couplingmedium to the vessel wall to heat the vessel wall and increase thevessel wall's temperature above a temperature of the liquid sample; andsimultaneous with applying acoustic energy to heat the vessel wall,applying acoustic energy from the acoustic energy source to the liquidsample to disrupt a boundary layer of the liquid sample at the vesselwall so as to enhance heat transfer from the vessel wall to the liquidsample and to raise the temperature of the liquid sample above atemperature of the coupling medium, heating of the liquid sample beingperformed at a rate of at least about 25 degrees C. per ml per minute.22. The method of claim 21, wherein the coupling medium that couplesacoustic energy from the acoustic energy source to the vessel wall is aliquid.
 23. The method of claim 21, wherein the coupling medium is at atemperature that is lower than a temperature of the liquid sample. 24.The method of claim 21, further comprising: subsequent to heating theliquid sample to a temperature greater than the temperature of thecoupling medium, stopping heating of the vessel wall to cool the vesselwall, and applying acoustic energy to the liquid sample to disrupt aboundary layer of the liquid sample at the vessel wall so as to enhanceheat transfer from the vessel wall to the liquid sample and to cool theliquid sample.
 25. The method of claim 21, wherein the coupling mediumincludes liquid water and the acoustic energy source includes atransducer in contact with the liquid water.
 26. The method of claim 21,wherein the acoustic energy is focused to form a focal zone of acousticenergy that is located at least in part at the vessel wall.
 27. Themethod of claim 21, wherein the acoustic energy is focused to form afocal zone of acoustic energy that is located inside the vessel.
 28. Themethod of claim 21, wherein the liquid sample increases in temperatureat a rate of at least 50 degrees C. per ml per minute.
 29. The method ofclaim 21, further comprising: subsequent to raising the temperature ofthe liquid sample above a temperature of the coupling medium,transferring heat from the vessel wall to the coupling medium via theheat exchanger so as to lower the temperature of the vessel wall; andsubsequent to raising the temperature of the liquid sample, applyingacoustic energy from the acoustic energy source to the liquid sample todisrupt the boundary layer of the liquid sample at the vessel wall totransfer heat from the liquid sample to the vessel wall and lower thetemperature of the liquid sample.
 30. A vessel for holding a liquidsample to be treated with acoustic energy, comprising: a vessel having awall with an inner surface and an outer surface and defining an interiorvolume to hold a liquid sample; and a heat exchanger feature at theinner surface of the wall, the heat exchanger feature including physicalstructure that disrupts a boundary layer of the liquid sample at thevessel wall in response to sample movement caused by acoustic energyapplied to the sample.
 31. The vessel of claim 30, wherein the heatexchanger feature includes a plurality of raised areas on the vesselwall.
 32. The vessel of claim 30, further comprising a heat exchangerfeature on the outer surface of the wall.
 33. The vessel of claim 30,wherein the interior volume is between 1 μL and 100 milliliters.